Stroke is a major acute neurological insult that disrupts brain function and causes neuron death. Recovery of lost function can occur after stroke and is attributed to brain reorganization and neuroplasticity. In this proposal, we will use optogenetics and imaging techniques to study brain circuit dynamics and axonal plasticity during post-stroke recovery. Optogenetics allows activation or inhibition of specific cell groups and circuits in the brain with millisecond-scale precision, thus is a valuable tool for studying neural circuits involved in post-stroke recovery. We recently demonstrated that selective neuronal stimulation in the ipsilesional motor cortex (iM1) using optogenetics can activate plasticity mechanisms and promote recovery. In Aim 1 we will use optogenetic functional MRI (ofMRI) to study global changes in brain circuit activation evoked by selective optogenetic stimulations during post-stroke recovery in non-stimulated stroke mice and repeatedly-stimulated stroke mice. In Aim 2A we will examine brain circuit activation at the cellular level in 3-D whole brain using two lines of activity reporter mice: neuronal activation reporter mice (FosTRAP) and synaptic activity reporter mice (ArcTRAP). We will determine the cell type of these activated neurons (excitatory vs inhibitory). In Aim 2B we will investigate how stimulations alter structural plasticity through axonal sprouting using the anterograde tracer (biotinylated-labeled dextranamine). The CLARITY technique will be used for Aim 2 to visualize cellular resolution of circuit activities and axonal sprouting in 3-D whole brains. In Aim 3 we will use the optogenetics technique to determine the role of the contralesional cortex (side opposite to stroke) during post-stroke recovery. Some studies indicate that contralesional cortex activation is necessary for recovery, whereas other studies suggest that contralesional cortex activation may be maladaptive and worsen recovery. To determine whether the contralesional cortex exerts beneficial or deleterious effects during recovery and whether there is a time-dependent role, we will manipulate specific neural circuits in the contralesional cortex at different post- stroke phases (early vs late) and examine their effects on functional recovery. Functional recovery will be evaluated by a panel of sensorimotor behavior tests that are well established in our lab. Activity-dependent neurotrophin expression and structural plasticity (axonal sprouting) will be examined in the groups that exhibit functional recovery. Our findings will advance the understanding of neural circuits and brain reorganization during post-stroke recovery.